Compositions normalizing circadian rhythm

ABSTRACT

The present invention provides a circadian rhythm normalizing composition containing astaxanthin and/or its ester as an active ingredient, and a composition having the action of enhancing the circadian rhythm normalizing action of melatonin by protecting melatonin. The composition can be in the form of a drug, a functional food, a food or a beverage. 
     This composition has a circadian rhythm normalizing action, and has the action of preventing or alleviating sleep disorder and various diseases due to disturbance of the circadian rhythm.

This application is the national phase of international applicationPCT/JP01/04027 filed 15 May 2001 which designated the U.S. which claimsforeign priority of JP143039/2000, filed May 16, 2000.

TECHNICAL FIELD

This invention relates to a circadian rhythm normalizing compositioncontaining astaxanthin and/or its ester as an active ingredient, and acomposition having astaxanthin and/or its ester incorporated in orcombined with melatonin in order to enhance the somniferous action ofmelatonin. The invention also relates to a composition incorporatingastaxanthin and/or its ester in order to enhance the somniferous actionof melatonin already present in vivo.

BACKGROUND ART

Living beings on the Earth have a mechanism, called a biological clock,for a circadian rhythm tuned to a 24-hour environmental cycle from thedaytime to the nighttime, such as a light-dark cycle, occurring as theEarth revolves on its own axis. The biological clock is controlled bythe hypothalamic suprachiasmatic nucleus of the brain. We have thecircadian rhythm of sleep-wakefulness and eating behavior attributed tothe biological clock.

The circadian rhythm is observed not only in sleep and diet, but also inmost of the body temperature, blood pressure, heart beat, and internalsecretion. Understanding the circadian rhythms of the autonomic nervoussystem, endocrine system, hemodynamics, etc and keeping them normal areconsidered important to the treatment and prevention of cardiovasculardiseases. In modern society, artificial and irregular lives due to workshifts, long distance jet flights, aging of the society, diverse lifestyles, and so on are becoming routine. In this modern society, thereare rapid increases in various diseases attributed to biorhythmdisorders, including circadian rhythm-associated sleep disorder.Effective therapeutic action against such diseases is urgently needed. Apatient with desynchronosis syndrome (time zone fatigue) due todisturbance of the circadian rhythm falls into insomnia at night, andsuffers from a sleep attack during the daytime. The patient is alsoannoyed by headache, tinnitus, palpitation, nausea, abdominal pain, ordiarrhea, and presents with symptoms of decline in judgment and decreasein concentration. Draw-back sleep phase syndrome (DSPS) shows thehighest morbidity rate during puberty (Diagnostic ClassificationSteering Committee, Thorpy M. J.: International Classification of SleepDisorders: Diagnostic and Coding Manual., American Sleep DisordersAssociation, Rochester. 1990). Such circadian rhythm-associated sleepdisorder among young people makes social adaptation difficult, andhinders the patients from exhibiting their abilities (Kajimura et al.:The Japanese Journal of Clinical Medicine, Vol. 56, No. 2, p. 404,1998).

Among methods for regulating the disturbance of the circadian rhythmproducing such symptoms are psychotherapeutic approaches,non-pharmacotherapies, and pharmacotherapies. The non-pharmacotherapiesinclude irradiation with high illuminance light, which nowadays isactively used for treatment of seasonal affective disorder and circadianrhythmical sleep disorder. For pharmacotherapies, vitamin B₁₂,benzodiazepine hypnotics, etc. are used, but they do not take effectwithout fail, and established pharmacotherapy is expected. Compared withthe hitherto predominant barbiturate or non-barbiturate hypnotics,benzodiazepine hypnotics minimally cause drug tolerance or dependence,and have relatively high safety. However, the benzodiazepine hypnoticsare known to produce adverse reactions, such as muscle relaxant action,carry-over effect, and amnestic action, and their use requires due care.That is, there are currently not any excellent compounds which are safe,effective and possessive of a circadian rhythm normalizing action.

Melatonin is expected as a new compound for regulating the circadianrhythm. Melatonin is a hormone mainly produced by the pineal body, andits production shows marked diurnal fluctuations. Its amount produced inthe nighttime reaches as large as 50 to 100 times that during thedaytime.

When the nocturnal secretion of melatonin is suppressed by theadministration of a β-blocker, there occur sleep-wakefulness rhythmdisorders, for example, a low quality sleep such as increased midwaywakening at night (Brismar et al.: Acta. Med. Scand., 223, p. 525, 1988)and a decreased wakefulness level during the daytime (Dimenas et al.: J.Clin. Pharmacol., 30, s103, 1990). Behind sleep disorder in elderlypeople lies a decrease in melatonin secretion, and melatonin replacementtherapy is considered effective for this disorder (Garfinkel et al.:Lancet, 346, p. 541, 1995). Of aged persons with decreased physiologicalsection of melatonin, those who can sleep well have a large amount ofmelatonin secreted (Haimov et al.: Sleep. 18, p. 598, 1995). Based onthese facts, endogenous melatonin is regarded as a physiologicalhypnotic substance which takes part in the regulation of thesleep-wakefulness rhythm.

Concerning the hypnotic action of exogenous melatonin, varieties ofresults have been reported. A report says that the administration ofmelatonin resulted in upgrading of sleep, such as shortening ofhypnagogic latency, decrease in halfway waking, or improvement ofinsomnia (Zhdanova et al.: Clin. Pharmacol. Ther., 57, p. 552, 1995). Onthe other hand, there is a report which denies the sleep regulatingeffect or the sleep disorder improving effect of melatonin (James etal.: Neuropsychopharmacology, 3, p. 19, 1990).

As one of the reasons why these contradictory results were obtained,dosing time dependency has been indicated (Mishima: The Japanese Journalof Clinical Medicine, Vol. 56, No. 2, p. 302, 1998). Another reason maybe the fact that melatonin is easily oxidized. In vivo melatonin isknown to be metabolized and deactivated by the oxidative ring cleavagereaction of the indole ring by indoleamine-2,3-dioxygenase in thepresence of superoxide anions (Hayaishi: J. Biochem., 79, p. 13, 1976).Thus, this reaction easily proceeds in aged persons in whom, or understress under which, an active oxygen or radical concentration, a causefor occurrence of superoxide anions, is said to be high. Eventually, theconcentration of melatonin may lower, making regulation of sleepdifficult.

As discussed above, melatonin is clearly a factor involved in theregulation of the circadian rhythm, but much remains to be solved forthe full performance of its therapeutic effect. Accordingly, if acircadian rhythm normalizing action can be found in other naturallyoccurring compounds, their value is very high for use in the preventionof sleep disorder and its associated various diseases. If a compoundcapable of raising the bioavailability of endogenous melatonin andexogenous melatonin safely and effectively is obtained from a naturalsubstance, its value in use is extremely high.

Astaxanthin is a red carotenoid pigment seen in aquatic animals such ascrustaceans or fish, and microorganisms. Astaxanthin has been used as abody color improver for fish, or as an additive for improvement of thecolor tone of domestic animals (Japanese Unexamined Patent PublicationNo. 206342/82, Japanese Unexamined Patent Publication No. 54647/85, andJapanese Unexamined Patent Publication No. 63552/92). The antioxidantaction of astaxanthin has been demonstrated to be more potent than thatof α-tocopherol, so that astaxanthin is expected to find use as ananti-oxidizing agent, a health food, a cosmetic, and a drug, in additionto application as a natural pigment (Eiji Yamashita: Foods andDevelopment, vol. 27, No. 3, p. 38, 1992). Recently, it has beenreported that astaxanthin, administered at a dose 1/100 the dose ofα-tocopherol, can suppress increases in lipid peroxides in the braincaused by irradiation with ⁶⁰Co (Nishigaki et al.: J. Clin. Biochem.Nutr., 16, p. 161, 1994).

However, it has not been known that astaxanthin has the action ofnormalizing the circadian rhythm, and is effective for preventing oralleviating sleep disorder and various diseases due to disturbance ofthe circadian rhythm. Nor is astaxanthin known to enhance the circadianrhythm regulating action of melatonin. Furthermore, it has not beenknown thus far to use astaxanthin as a food or beverage, a foodadditive, or an active ingredient of a drug, intended for a circadianrhythm normalizing effect and a hypnotic action.

DISCLOSURE OF THE INVENTION

An object to be attained by the present invention is to provide acomposition, which is highly safe, which regulates the circadian rhythmeffectively, and which is aimed at preventing or alleviating medicalsymptoms of sleep disorder and various diseases due to disturbance ofthe circadian rhythm, and a food, beverage or drug containing thecomposition.

Another object of the invention is to provide a composition, which actsto increase the availability of melatonin in vivo, effectivelyregulating the circadian rhythm, and a food, beverage or drug containingthe composition.

To search for a substance having the action of normalizing the circadianrhythm, the inventors of the present invention conducted studies onvarious substances with the use of a circadian rhythm measuring method,with the amount of spontaneous movement of rats as an indicator, in anenvironment controlled to have a 12-hour light period and a 12-hour darkperiod. As a result, the inventors have clarified that astaxanthin,which is one of carotenoids, and/or its ester has a circadian rhythmnormalizing action. Further, they have found that the circadian rhythmnormalizing action of melatonin can be enhanced by ingesting astaxanthinand/or its ester and melatonin in combination. These findings led themto accomplish the present invention.

The present invention provides a composition, such as a drug or a food,which contains astaxanthin and/or its ester as an active ingredient, orwhich contains melatonin, astaxanthin and/or its ester so as to beingestible in combination. Such a combination is highly safe, and showsa circadian rhythm normalizing action.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a view showing the influence of astaxanthin on the totalamount of movements (day-to-day changes).

FIG. 2 is a view showing the influence of astaxanthin on the ratio ofmovements in the light period to the total amount of movements(day-to-day changes).

FIG. 3 is a view showing the influence of astaxanthin on the ratio ofmovements in the light period to the total amount of movements(average).

FIG. 4 is a view showing the influences of astaxanthin and melatonin onthe total amount of movements (averages).

FIG. 5 is a view showing the influences of astaxanthin and melatonin onthe ratio of movements in the light period (averages).

FIG. 6 is a view showing the effect of low-dose α-tocopherol on thecircadian rhythm normalizing action of melatonin.

FIG. 7 is a view showing the effect of high-dose α-tocopherol on thecircadian rhythm normalizing action of melatonin.

FIG. 8 is a view showing the effect of astaxanthin on the circadianrhythm normalizing action of melatonin.

FIG. 9 is a view showing the effect (dose dependency) of astaxanthin onthe ratio of movements in the light period (averages).

FIG. 10 is a view showing the effect (dose dependency) of astaxanthin onthe total amount of movements (averages).

EMBODIMENTS OF THE INVENTION

The present invention made it possible to search for a substance havinga circadian rhythm normalizing action with the use of a circadian rhythmmeasuring method, with the amount of spontaneous movements of ratsreared in a controlled environment being used as an indicator. As aresult, astaxanthin, which is one of carotenoids, and/or its ester hasbeen found to have a circadian rhythm normalizing action. That is, bytaking astaxanthin into rats with a disturbed circadian rhythm, thecircadian rhythm of the rats was successfully brought close to thenormal value.

Further, the present invention has found that the circadian rhythmnormalizing action of melatonin can be enhanced by ingesting astaxanthinand/or its ester and melatonin simultaneously. This enhancive action wasnot observed in α-tocopherol, an antioxidant like astaxanthin. Thisaction of astaxanthin has been attributed to the antioxidant action ofastaxanthin, because melatonin is a compound susceptible to oxidativedecomposition. In other words, the effect of enhancing the circadianrhythm normalizing action of melatonin has been assumed to be an actioncommon to anti-oxidizing substances. Thus. α-tocopherol, an antioxidantlike astaxanthin, was used instead of astaxanthin to compare the effectsof α-tocopherol and astaxanthin (Example 3). As a result, 0.001% and0.005% of α-tocopherol showed no effect of enhancing the circadianrhythm normalizing action of melatonin. On the other hand, 0.0045% ofastaxanthin was found to enhance the circadian rhythm normalizing actionof melatonin significantly. This has not been known as the action ofastaxanthin.

The astaxanthin and/or its ester of the present invention can takevarious forms, and can be supplied as foods, such as common foods,health foods, functional foods, and nutrient supplement foods, or drugs.The amount of melatonin used when melatonin is jointly used may besuitably increased or decreased according to the amount necessary toexhibit the function of melatonin. In the composition of the presentinvention, its dose may be as low as 50 to 80% of the dose so farrequired, since the availability of melatonin in vivo is improved in thecomposition.

The astaxanthin and/or its ester, the active ingredient of the presentinvention, has been discovered from the crust and egg of crustaceans(Kuhn et al.: Angew. Chem., 51, p. 465, 1938 or Ber., 71, p. 1879,1938), organs (Kuhn et al.: Ber. 72, p. 1879, 1939), plants (Tischer etal.: Z. Physiol. Chem., 267, p. 281, 1941), petals of Amur Adonis orbuttercup (Seybold et al.: Nature, 184, p. 1714, 1959), skin of variousfishes and shellfishes (Matsuno et al.: Carotenoids Chemistry andBiology, Plenum Press, p. 59, 1989), egg (Miki et al.: Comp. Biochem.Physiol., 71B, p.7, 1982), Antarctic krills (Yamaguchi et al.: Bull.Japan. Soc. Sci. Fish, 49, p. 1411, 1983), Haematococcus ofChlorophyceae (Renstroem et al.: Phytochem. 20, p. 2561, 1981), redyeast Phaffia (Andrewes et al.: Phytochem., 15, p. 1003, 1976), andmarine bacterium Agrobacterium aurantiacum (Yokoyama et al.: Biosci.Biotech. Biochem., 58, p. 1842, 1994). The chemical structure ofastaxanthin has been determined (Andrewes et al.: Acta Chem. Scand.,B28, p. 730, 1974), and a method for its organic synthesis has also beenestablished (Widmer et al.: Helv. Chem. Acta., 64, p. 2405, 1981, Mayeret al.: Helv. Chem. Acta., 64, p. 2419, 1981). Thus, astaxanthin can beeasily obtained as a chemical synthetic product.

The active ingredient in the present invention may be chemicallysynthesized astaxanthin, or may be an extract from the crust ofcrustaceans such as red yeast, Tigriopus (red water flea) or krills, orgreen algae, or minute algae (may be in the state of a solvent extract,or if desired, a suitably purified material) which contains astaxanthinand/or its ester. Alternatively, there can be used a powder formed bypulverizing crustaceans or green algae of the genus Haematococcuscontaining astaxanthin and/or its ester.

For example, the following are cited as methods for preparation ofastaxanthin and/or its ester: methods for preparation utilizingcrustaceans (Japanese Unexamined Patent Publication Nos. 1983-88353 and1989-186860), methods for preparation utilizing algae (JapaneseUnexamined Patent Publication No. 1989-187082, Officially PublishedPatent Gazette No. 1990-501189, Japanese Unexamined Patent PublicationNo. 1993-68585, Japanese Unexamined Patent Publication No. 1991-83577),methods for preparation utilizing astaxanthin-producing yeast(Officially Published Patent Gazette No. 1990-504101, JapaneseUnexamined Patent Publication No. 1991-206880, Japanese UnexaminedPatent Publication No. 1992-228064), and methods for preparationutilizing krills (Japanese Unexamined Patent Publication No. 1985-4558,Japanese Unexamined Patent Publication No. 1986-281159, Eiji Yamashita:Foods and Development, vol. 27, p. 38(1992)). Novel synthesis ofastaxanthin (Japanese Unexamined Patent Publication No. 1992-225933) isalso disclosed. Other methods can be used alone or in combination, ifthey enable the properties of the active ingredient of the presentinvention to be effectively utilized.

The ester of astaxanthin used in the present invention is an ester ofastaxanthin with an arbitrary fatty acid acceptable for foods ormedicines. Examples of the fatty acid are saturated fatty acids, such aspalmitic acid and stearic acid, or unsaturated fatty acids such as oleicacid, linoleic acid, α-linolenic acid, γ-linolenic acid,bishomo-γ-linolenic acid, arachidonic acid, eicosapentaenoic acid, anddocosahexaenoic acid. Any monoesters or diesters comprising one or twoof them joined to astaxanthin can be used in the present invention.

When the active ingredient of the present invention is used as a food ordrug, any of the above-described crude extracts or purified products canbe used. When any of them is used, the active ingredient can beemulsified according to the customary method, or can be emulsified withthe addition of a compound which will be a synergist, becauseastaxanthin and its ester are oily.

Astaxanthin and/or its ester, the active ingredient of the food,beverage or drug of the present invention, is contained in such aneffective amount, for example, of 0.5 to 500 mg/day, preferably 5 to 50mg/day, as will exhibit a circadian rhythm normalizing action when thefood, beverage or drug is taken into the body. However, there is norestriction on the upper limit of the active ingredient.

Melatonin in the present invention, on the other hand, may be asynthetic product, or may be an extract from an animal or plantcontaining melatonin, or a purified product or a crude extract. Any ofthem can be used without any restriction.

Astaxanthin and/or its ester can be added to or combined with melatoninin a varying ratio, and ingested. To exhibit the effect of the presentinvention, the amount of astaxanthin and/or its ester is desirably 3 to6 times the weight of melatonin.

For the composition or preparation of the food, beverage or drug of thepresent invention, astaxanthin and/or its ester as the active ingredientof the present invention may be a chemically synthesized product, or anaturally occurring substance. They can be used singly or in a suitablecombination. Astaxanthin or the crude extract can be dissolved inethanol, and used as such or after dilution with water.

If desired, an emulsified preparation can be produced. To produce theemulsified preparation, gallic acid, L-ascorbic acid (or its ester orsalt), gum (such as locust bean gum, gum Arabic, or gelatin), andfurther vitamin P (e.g., flavonoids or polyphenols such as hesperidin,rutin, quercetin, catechin, and cyanidin, or mixtures thereof) can beadded to the aqueous phase. To the oil phase, astaxanthin or its ester,the crude extract, or a mixture thereof can be added. Further, glycerol,glycerin fatty acid ester, sorbitan fatty acid ester, sucrose fatty acidester, dextrin, or oils and fats, such as ordinary liquid oils, e.g.,rapeseed oil, soybean oil, or corn oil, can be added. These materialsare emulsified, whereby the emulsified preparation can be easilyprepared. For emulsification, a high speed agitator, a homogenizer orthe like can be used to perform emulsification with mixing.

The active ingredient as the drug of the present invention is orallyadministered as a solid dosage form such as tablets or powder, or aliquid dosage form such as an elixir, a syrup or a suspension.Parenterally, the drug can be used, for example, as an injection orsuppositories. Adjuvants for use in the oral drug include, for example,carriers in the form of solid powders, sugars such as lactose andsaccharose, amino acids such as glycine, and cellulose. As lubricants,silicon dioxide, talc, magnesium stearate, and polyethylene glycol canbe exemplified. As binders, starch, gelatin, methyl cellulose, andpolyvinyl pyrrolidone can be cited. Disintegrants include starch andagar.

The composition of the present invention has the action of normalizingthe circadian rhythm, and so can be used for the purpose of preventionor alleviation of circadian rhythmical sleep disorder. The compositioncan also be used for normalizing not only the rhythm of sleep, but alsothe rhythms of hormone secretion, nervous activity, enzyme activity,metabolism, body temperature, renal function, etc. which involvecircadian rhythms. Hence, the composition can prevent or alleviatemedical symptoms of various diseases associated with the abnormality ofthese rhythms. Alleviation or improvement of the symptoms in the presentinvention include the treatment of diseases.

Moreover, the astaxanthin of the present invention has the effect ofenhancing the circadian rhythm normalizing action of melatonin.Surprisingly, this action was not noted in the same dose ofα-tocopherol, an antioxidant. Hence, the mere fact that astaxanthin isan antioxidant cannot explain the action and effect of astaxanthin thatenhances the circadian rhythm normalizing action of melatonin.

The amount of astaxanthin or its ester (active ingredient) contained inthe drug of the present invention is such an amount as can be orally orparenterally administered in a dose of normally 0.5 to 500 mg,preferably 5 to 50 mg, daily in an adult. The dose clearly differsaccording to the type of disease for which the astaxanthin or its esteris administered, the age of the patient, the body weight of the patient,the severity of symptoms, or the dosage form. The active ingredient ofthe present invention has a circadian rhythm normalizing action, andthus can prevent or improve sleep disorder and various diseases, such ascardiovascular disorders, which result from the disturbance of thecircadian rhythm.

The food or beverage of the present invention can be supplied in theform of common foods, including margarine, butter, butter sauce, cheese(natural cheese, processed cheese), fresh cream, shortening, lard, icecream, yogurt, coffee milk, dairy products, sauce, soup, meat products,fish products, popcorn, fried potatoes, potato chips, seasoned finelydivided foods (e.g., sesame, seaweed, fish meat, egg, and egg) forsprinkling over rice, rolled omelet sometimes containing fish meatpaste, Japanese confectionery (rice cracker, etc.), Westernconfectionery (custard pudding, jelly, gummy candy, candy, drop,caramel, chocolate, chewing gum, pastry, etc.), baked confectionery(sponge cake, cakes, doughnut, biscuit, cookie, cracker, etc.),macaroni, pasta, salad oil, instant soup, dressing, egg, mayonnaise,miso, non-alcoholic beverages such as carbonated beverages,non-carbonated beverages (fruit juice drink, nectar, etc.), soft drinks,sport drinks, tea, coffee, and cocoa, and alcoholic beverages such asliqueur and medicated liquor.

The food or beverage of the present invention can be produced byblending astaxanthin and/or its ester or a product, which has beenobtained from a natural substance containing astaxanthin and/or itsester, with raw materials for an ordinary food, and processing themixture by an ordinary method. The concentration of astaxanthin and/orits ester blended differs according to the dosage form or the shape orproperties of the food. Generally, the concentration is preferably, butnot restricted to, 0.001 to 10%. However, the food or beverage isprepared such that the active ingredient of the present invention, asastaxanthin, is contained in the necessary amount, per daily intake ofthe final product, to show a circadian rhythm normalizing action. As faras is known currently, the preferred amount is 0.5 to 500 mg, preferably5 to 50 mg, per daily intake of the product in the adult. Such an amountcan be selected, as desired, by people skilled in the art.

When the food or beverage of the present invention is used as a nutrientsupplement food or a functional food, its form may be the same form asthe aforementioned pharmaceutical preparation. However, the food orbeverage may be in a processed form, such as a liquid natural food, apartially digested nutritional diet, an elemental diet, an ampuledliquid medicine type product, capsules, or an enteral nutrition, whichcontains, for example, protein (protein of a high nutritional value andhaving a good amino acid balance, such as milk protein, soybean protein,or ovalbumin, is used most widely as the source of protein, but thedecomposition product thereof, the oligopeptide of egg white, thehydrolysis product of soybean, or a mixture of them with a single aminoacid is also used), sugars, fat, trace elements, vitamins, emulsifier,and flavors. When the food or beverage is supplied as a sport drink or anutritional drink, easily digestible carbohydrates, nutritionaladditives such as amino acids, vitamins and minerals, sweeteners,spices, flavors, and pigments can be incorporated in order to recoverthe nutritional balance and better the flavor of the food or beveragewhen ingested.

The nutrient supplement food or functional food of the present inventionis not restricted to them, and may be in the form of ordinary foods asdescribed above. If possible, however, it should desirably be in a unitintake form.

The Examples and Referential Examples will be described in detail below,but the present invention is not limited thereby.

EXAMPLE 1 Circadian Rhythm Normalizing Action of Astaxanthin

The influence of astaxanthin on daily movements was investigated inrats. Rats have the habit of resting (sleeping) mainly in the lightperiod, and acting in the dark period. The circadian rhythm of rats wasinvestigated by measuring the amount of daily movements continuously,and examining changes over time in the amounts of movements in the lightperiod and the dark period.

Four-week-old male Wistar rats (purchased from Nippon Charlesriver) werepreliminarily reared for one week, and subjected to experiments at theage of 5 weeks. The rats were randomly divided into two groups (5 to 6rats/group), and one of the groups was used as a control diet (lowtocopherol) group, while the other group was used as an astaxanthin dietgroup, each group receiving the designated diet for 4 weeks. The animalswere reared in animal cages kept at room temperature of 23.5±1° C. and ahumidity of 55±5%, and the feed and water were provided ad libitum.

Methods for Measuring the Amount of Daily Movements in Rats

Formulation of Feed

The feed was prepared according to the formulation shown in Table 1.Astaxanthin used was astaxanthin extracted from krills and then purified(an oil containing 15% of astaxanthin as a free compound; a product ofItano Refrigerating Co.).

Measurement of the Amount of Rat Daily Movements

The rats were individually reared in a box equipped with an infraredsensor, and the amount of movements was counted with the infraredsensor. That is, each time the animal passed the infrared sensor, thepassage was taken as one count. The counts taken were accumulated, andexpressed as the amount of movements (counts). Statistical treatment wasperformed using ANOVA and student t-test.

A period of 1 week after start of rearing with the experimental diet wasset as a tuning period in which the animal was tuned to a 12-hour darkperiod and a 12-hour light period. Then, from the 2nd week, the amountof daily movements was continuously measured for 3 weeks. The totalamount of movements, and the ratio of movements in the light period tothe total amount of movements (i.e., the amount of movements in thelight period/the total amount of movements×100 (%)) were calculatedevery day. Astaxanthin did not affect the total amount of movements incomparison with the control (FIG. 1), but lowered the ratio of movementsin the light period (FIG. 2). Based on these findings, the ingestion ofastaxanthin was found to decrease the activity of rats in the lightperiod, and astaxanthin was confirmed to have the action of stabilizingthe circadian rhythm. The averages of the ratio (%) of movements in thelight period were compared in both groups (FIG. 3). A significantdecrease in the ratio of movements in the light period was observed inthe astaxanthin diet group, demonstrating astaxanthin to have the actionof normalizing the circadian rhythm.

EXAMPLE 2 Enhancive Effect of Astaxanthin on Circadian RhythmNormalization by Melatonin

Four-week-old male Wistar rats (purchased from Nippon Charlesriver) werepreliminarily reared for one week, and subjected to experiments at theage of 5 weeks. The rats were randomly divided into two groups (10 to 11rats/group), and the two groups were given a control diet (lowtocopherol diet) and an astaxanthin diet (Table 1), respectively. Eachgroup was further divided into two groups (5 to 6 rats/group), whichreceived distilled water and melatonin water, respectively, for 4 weeks.The animals were reared in animal cages kept at room temperature of23.5±1° C. and a humidity of 55±5%, and the feed and water were providedad libitum. Melatonin was dissolved in water at a concentration of 0.5mg/100 ml, and the solution was given ad lib, as drinking water. Theintake of melatonin, calculated from the amount of water taken by rats,was about 1 mg/day.

After start of rearing with the experimental diet, a tuning period of 1week was provided. Then, the amount of movements of rats was measured asin Example 1 for 3 weeks to look into changes in the circadian rhythm.No differences were noted among the averages of the total amount ofmovements in the respective groups (FIG. 4). The ratio of activity inthe light period to the total amount of movements (the amount ofmovements in the light period/the total amount of movements×100 (%)) wascompared in the respective groups. A tendency toward a decrease in theratio of activity in the light period was seen even in the astaxanthingroup or melatonin group alone as compared with the control group.Astaxanthin and melatonin administered simultaneously further enhancedthe circadian rhythm normalizing action (FIG. 5).

EXAMPLE 3 Comparison with the Enhancive Effect of α-Tocopherol onCircadian Rhythm Normalization by Melatonin

The influence that α-tocopherol, a liposoluble antioxidant likeastaxanthin, exerts on the circadian rhythm normalizing action ofmelatonin was compared with that of astaxanthin.

Four-week-old male Wistar rats (purchased from Nippon Charlesriver) werepreliminarily reared for one week, and subjected to experiments at theage of 5 weeks. The rats were randomly divided into two groups (10 to 11rats/group), and each group was given 0.001% and 0.005% tocopheroldiets. Each group was further divided into two groups (5 to 6rats/group), and one of the groups received distilled water for 4 weeks,while the other group received melatonin water for 4 weeks. The animalswere reared in animal cages kept at room temperature of 23.5±1° C. and ahumidity of 55±5%, and the feed and water were provided ad libitum.

After start of rearing with the experimental diet, a tuning period of 1week was provided. Then, the amount of movements of rats was measuredfor 3 weeks in the same manner as in Example 1 to look into changes inthe circadian rhythm. Day-to-day changes in the ratio of movements inthe light period to the total amount of movements (i.e., the amount ofmovements in the light period/the total amount of movements×100 (%)) areshown in FIGS. 6, 7 and 8. An enhancive effect on the circadian rhythmnormalizing action of melatonin was not observed in the 0.001%tocopherol diet group (FIG. 6). A slight enhancive effect on thecircadian rhythm normalizing action of melatonin was observed in the0.005% tocopherol diet group (FIG. 7). However, the enhancive effect onthe circadian rhythm normalizing action of melatonin, which was observedin the 0.005% tocopherol diet group, was weaker than the enhanciveeffect on the circadian rhythm normalizing action of melatonin that wasobserved in the 0.0045% astaxanthin diet group (FIG. 8). These resultsclarify that astaxanthin has a more potent effect of enhancing thecircadian rhythm normalizing action of melatonin than does α-tocopherol.

EXAMPLE 4 Dose Dependency of the Circadian Rhythm Normalizing Action ofAstaxanthin

Four-week-old male Wistar rats (purchased from Nippon Charlesriver Co.)were preliminarily reared for one week, and subjected to experiments atthe age of 5 weeks. The rats were randomly divided into five groups,which were given a control diet (tocopherol-deficient diet) andastaxanthin diets (Table 2), respectively. The feed and water wereprovided ad libitum. The amount of astaxanthin intake calculated fromthe amount of the feed taken into the rats was about 2 to 20 mg/day.

After a tuning period of 1 week, the amount of movements of rats wasmeasured for 3 weeks in the same manner as in Example 1 to investigatechanges in the circadian rhythm. The ratio of activity in the lightperiod to the total amount of movements (i.e., the amount of movementsin the light period/the total amount of movements×100 (%)) decreased inan astaxanthin concentration-dependent manner, starting with a minimumdose of 0.15 g/kg diet. The decrease was significant at doses of 0.3g/kg diet and more (FIG. 9). The total amount of movements, on the otherhand, tended to increase at a maximum dose of 1.5 g/kg diet (FIG. 10).

The present example used the tocopherol-deficient diet as the basicfeed. As in the case of Example 1, in which the low tocopherol diet wasused as the basic feed, the circadian rhythm normalizing action ofastaxanthin was confirmed in the present example. Thus, astaxanthin wasconfirmed to act effectively even when tocopherol was deficient todisturb the circadian rhythm.

[Preparation Example 1] Emulsified preparation (% by weight) Oil phaseAstaxanthin 0.1 Rapeseed oil 39.9 Succinic acid glyceride 2.0 Aqueousphase L-Ascorbic acid 2.0 Gallic acid 1.0 Quercetin 1.0 Locust beam gum0.1 Water 53.9

Water having locust beam gum dissolved therein was heated to 65° C., andthen mixed with gallic acid, L-ascorbic acid and quercetin. The oilphase, which had been formed by mixing and dissolution at 65° C. wasmixed with the aqueous phase with stirring. The mixture was passedthrough a homogenizer for homogenization, and cooled to 10° C. to obtainan emulsified preparation of the above formulation. This emulsifiedpreparation is drunk in an amount of about 1 to 20 ml per dose.

[Preparation Example 2] Emulsified preparation (% by weight) Oil phaseAstaxanthin 0.1 Rapeseed oil 38.9 Citric acid monoglyceride 2.0 Aqueousphase L-Ascorbic acid 2.0 Gallic acid 1.0 Hesperidin 1.0 Locust beam gum0.05 Water 54.95

Water having locust beam gum dissolved therein was heated to 65° C., andthen mixed with gallic acid, L-ascorbic acid and hesperidin. The oilphase, which had been formed by mixing and dissolution at 65° C., wasmixed with the aqueous phase with stirring. The mixture was passedthrough a homogenizer for homogenization, and cooled to 10° C. to obtainan emulsified preparation of the above formulation. This emulsifiedpreparation is drunk in an amount of about 1 to 20 ml per dose.

[Preparation Example 3] Tablets (% by weight) Astaxanthin 5 Lactose 80Heavy magnesium oxide 15

The above materials were mixed uniformly, and formed into tablets with acontent of 180 mg/tablet.

[Preparation Example 4] Powder and granules (% by weight) Astaxanthin 45Lactose 40 Starch 15

The above materials were mixed uniformly, and formed into a powder orgranules.

[Preparation Example 5] Capsules (% by weight) Gelatin 70.0 Glycerin22.9 Methyl para-hydroxybenzoate 0.15 Propyl para-hydroxybenzoate 0.51Water Proper amount Total 100%

Soft capsule shells comprising the above components were charged with akrill extract fat (containing 1.7% astaxanthin diester) by the usualmethod to obtain soft capsules with a content of 180 mg/capsule.

[Preparation Example 6] Ampuled liquid medicine Taste: SodiumDL-tartrate 1 g Succinic acid 0.09 g Sweetness: Liquid sugar 8 KgSourness: Citric acid 120 g Vitamins: Vitamin C 100 g Vitamin E 300 gAstaxanthin ethyl ester 10 g Cyclodextrin 50 g Flavor 150 ml Potassiumchloride 10 g Magnesium sulfate 5 g

The above components were blended, and water was added to make a totalamount of 100 liters. The resulting ampuled liquid medicine is drunk inan amount of about 100 ml per dose.

[Preparation Example 7] Melatonin-containing tablets (% by weight)Astaxanthin  6 Melatonin  2 Vitamin B₆ 10 Calcium 30 Heavy magnesiumoxide 51

The above materials were mixed uniformly, and formed into tablets with acontent of 100 mg/tablet.

[Preparation Example 8] Melatonin-containing capsules (% by weight)Gelatin 70.0 Glycerin 22.9 Methyl para-hydroxybenzoate 0.15 Propylpara-hydroxybenzoate 0.51 Water Proper amount Total 100%

Soft capsule shells comprising the above components were charged with akrill extract fat (5% astaxanthin as a free compound) and melatonin bythe usual method to obtain soft capsules with a content of 180mg/capsule.

[Industrial Applicability]

The astaxanthin and/or its ester, contained in the food, beverage ordrug of the present invention, has a circadian rhythm normalizing actionas described in the Examples. The protective effect of astaxanthinand/or its ester on endogenous melatonin, or exogenous melatoniningested simultaneously with astaxanthin, enhances the efficacy ofmelatonin, acting to normalize the circadian rhythm. In the prior art,no substances having such an action have been known.

TABLE 1 Formulation of Experimental Feed Amount (g/kg) Component Controldiet Astaxanthin diet Casein (α-tocopherol free) 200 200 DL-methionine 33 Corn starch 150 150 Sucrose 500 500 Cellulose powder 50 50 Lard(α-tocopherol free) 50 50 Minerals (AIN-76) 35 35 Vitamins (AIN-76,α-tocopherol free) 10 10 Choline bitartrate 2 2 α-Tocopherol 0.01 0.01Astaxanthin* — 0.3 *Oil containing 15% astaxanthin as a free compound

TABLE 2 Formulation of Experimental Feed Amount (g/kg) Astaxanthin dietComponent Control diet 0.15 0.3 0.6 1.5 Casein (α-tocopherol free) 200200 200 200 200 DL-methionine 3 3 3 3 3 Corn starch 150 150 150 150 150Sucrose 500 500 500 500 500 Cellulose powder 50 50 50 50 50 Lard(α-tocopherol free) 50 50 50 50 50 Minerals (AIN-76) 35 35 35 35 35Vitamins (AIN-76, 10 10 10 10 10 α-tocopherol free) Choline bitartrate 22 2 2 2 Astaxanthin* — 0.15 0.3 0.6 1.5 *Oil containing 15% astaxanthinas a free compound

1. A method of normalizing circadian rhythm by administering to apatient suffering from disturbance of circadian rhythm, an effectiveamount of astaxanthin and/or an ester thereof, and an effective amountof melatonin.
 2. The method of claim 1 wherein said effective amount ofastaxanthin and/or an ester thereof ranges from 0.5–500 mg/day.
 3. Themethod of claim 1 wherein said effective amount of astaxanthin and/or anester thereof ranges from 5–50 mg/day.